Tanshinone IIA enhances the ovarian reserve and attenuates ovarian oxidative stress in aged mice

Abstract Background Tanshinone IIA (TSA), a major lipophilic component extracted from the roots of Salvia miltiorrhiza Bunge, has been widely used in China for its various biological activities. However, its effect on ovarian reserve in aged mice was not studied elsewhere. Objectives This study aimed to explore the effect of TSA on the ovarian reserve of aged mice as well as young mice. Forty weeks old mice (N = 40) were considered as aged group compared to 4 weeks old mice (N = 40), and these groups were subdivided into four subgroups (N = 10) to receive different doses of TSA (0, 10, 20, and 40 μg/g/day). Methods The effect of TSA was evaluated by counting follicular number by histological examination. Basal serum levels of FSH, LH, E2, and anti‐Mullerian hormone (AMH) were measured by ELISA. Moreover, the expression levels of antioxidant genes (CAT, Nrf2, GPX1), gap junction (Cx37), ERK1/2, and Smad5 family gene were examined at both mRNA (qPCR) and protein levels (western blot). Results Follicular number, level of AMH and E2, and the expression of CAT, Nrf2, and GPX1 genes increased significantly (p < 0.05) in aged mice administrated with medium (20 μg/g/day) and high (40 μg/g/day) doses of TSA, whereas FSH and LH levels were significantly low compared to low dose (10 μg/g/day) and control (0 μg/g/day) aged subgroups. However, we did not observe any effect of all doses of TSA on young mice. Conclusions Administration of TSA with medium and high doses up‐regulates the expression of antioxidative genes, reduces the oxidative injury, increases levels of AMH, and E2 levels that are relatively comparable to those in young mice, and consequently results in a healthy oocyte development.


INTRODUCTION
Female fertility is affected by multiple factors, including genetic, environmental, nutritional, and endocrinological factors. Ovarian function has the major role in maintaining a healthy reproductive function during female lifetime. A common feature in females of most mammalian species is age-related decline in fertility (Moussa et al., 2015).
There are two reasons for this: age-related follicular and endocrine changes (Malhi et al., 2005). Reproductive aging in cattle is associated with elevated circulating concentrations of gonadotropins, reduced concentrations of steroid hormones, and fewer 4-to 5-mm follicles were recruited into a wave; and, secondly, maternal age-related mitochondrial dysfunction (Takeo et al., 2012). This dysfunction is due to increased oxidative stress, deletions, or point mutations and variations in the mitochondrial genome (Shamsi et al., 2013;Takeo et al., 2012).
Ovarian reservation (OR) refers to the number and quality of oocytes, and it plays an essential role in achieving pregnancy. Diminished OR is a term used to denote that ovaries have eggs that are lower in number or quality than expected, and is characterized by increased apoptosis rate of granulosa cells (Y. Fan et al., 2019) and poor fertility outcomes (Chang et al., 2018). The condition may result from disease or injury, but most commonly occurs as a result of normal aging.
Delayed childbearing is the main challenge in reproductive medicine as increased age has an adverse effect on successful pregnancy, both in natural and assisted reproduction. With the continuous increase of age, the function of ovarian interstitial cells decreases, the proportion of granulosa cell apoptosis increases, and the number and quality of oocytes gradually decrease (Yuan et al., 2016). Unfortunately, there is no specific treatment that can slow ovarian aging and truly prevent diminished ovarian reserve.
Various pre-clinical and clinical studies have asserted the role of TSA in targeting tumour cells. Its anti-tumour mechanism may be involved in inhibiting tumour growth, inducing apoptosis, regulating cell cycle, regulating signalling pathways, and reversing the multidrug resistance in various human tumour cells (Kim et al., 2015). It also improves the activity of transcription factors of various antioxidant enzymes and reduces the oxidative stress response (Fu et al., 2007). The combination of TSA and other clinical commonly used drugs could enhance the therapeutic effect of these drugs, which is of great significance to the effective use of TSA in the future (Ansari et al., 2021).
The estimation of diminished ovarian reserve is routinely performed through measurement of hormone levels (Cohen et al., 2015). In this context, serum levels of follicle stimulating hormone (FSH), anti-Mullerian hormone (AMH), Luteinizing hormone (LH), and Oestradiol (E2) have been suggested as potential markers of fertility (Ramalho de Carvalho et al., 2012;Sharara et al., 1998), among which AMH is suggested as the most sensitive and specific available marker (Kaya et al., 2010;Visser et al., 2012). These hormone levels can show important information about how the ovaries and pituitary gland are working together.
To the best of our knowledge, the effect of TSA on ovarian reserve in aged mice was not studied elsewhere. The aim of the current study was to explore the effect of TSA on the ovarian reserve of aged mice. Fol-

Experimental animal preparation
The female Kunming mice used in the experiment were purchased from the Experimental Animal Centre. During the experiment, 4 weeks old mice were used as the control group (N = 40), and 40 weeks old mice were considered as the aged group (N = 40). Both groups were subdivided into four subgroups (N = 10) to receive intraperitoneal injection of TSA (Selleck, S2365) at different doses as follow: control (0 μg/gram body weight/day), low dose (10 μg/g/day), medium (20 μg/g/day), and high (40 μg/g/day) doses of TSA. After 2 weeks of daily injection, blood was collected by orbital blood sampling. The peripheral blood of the mouse was centrifuged to separate the serum, and it was frozen for later use. Mice were sacrificed and one ovary was fixed in 4% paraformaldehyde for histological examination, and the other ovary was placed in a cryotube and stored at −80 • C for RNA and protein extraction.

Histological examination
The ovarian tissue was fixed with 4% paraformaldehyde for 48 h, then repaired, dehydrated, and embedded, then cut into 5 μm sections, and stained with H&E staining kit (Solarbio, G1121

Quantitative real-time PCR
Six pools of ovarian tissues for each experimental group were formed.
Total RNA was extracted from each pool of by using Trizol® reagent  Table 1.

Western blot
Ovarian tissue was lysed in RIPA-PMSF protein extraction reagent (Invitrogen, 89900), homogenized at low temperature, and centrifuged to obtain a total protein lysate. After the concentration was determined by the bicinchoninic acid assay, the concentration of each histone was adjusted to be consistent. Lysates were diluted with 6 × protein loading buffer (Invitrogen, NP0007) and heated to 100

Statistical analysis
All data in this study were statistically analyzed using SPSS 22.0 software. The experimental test data are expressed in the form of standard mean deviation (mean ± SD), single-factor ANOVA is used for comparison between multiple groups, and SNK is used for pairwise comparison. p ≤ 0.05 indicates that the difference is statistically significant.

The effect of TSA on the number of follicles in ovarian tissue
Following administration of TSA, there was no significant (p > 0.05) difference between different doses of TSA in the number of ovarian follicles in the control group (35.4 ± 7.16, 33.6 ± 6.18, 34.7 ± 6.53, 31.5 ± 5.5), indicating that TSA has no effect on the ovarian reserve of young mice. However, administration of high and medium doses of TSA in aged group significantly (p < 0.05) increased the ovarian reserve (25.2 ± 3.52, 21.8 ± 6.32) compared to low dose (13.9 ± 4.28) and control aged subgroups (11.4 ± 4.15), respectively (Figure 1).

The relative abundance of mRNA of ERK1/2, Smad5, Nrf2, and GPX1 in the ovarian tissue of young and aged mice
The results showed that there was no significant difference between different doses of TSA on the expression level of ERK1/2, connexin37, F I G U R E 2 Hormonal level of anti-Mullerian hormone (AMH), E2, follicle stimulating hormone (FSH), and Luteinizing hormone (LH) in the serum of young (4 weeks old) and aged (40 weeks old) mice after administration of different doses of tanshinone IIA (TSA) (0, 10, 20, 40 μg/g/day). Different letters above the bars (a, b, c, and d) indicate significant differences (p < 0.05) CAT, Smad5, Nrf2, and GPX1 in the ovarian tissue of young mice. However, after administration of TSA in aged mice, particularly medium and high doses of TSA, the expression level of ERK1/2, connexin37, CAT, Smad5 and Nrf2 was significantly (p < 0.05) up-regulated com-pared with the control aged subgroup (Figure 3). The results of protein expression were consistent with that of mRNA. Such results support the major role played by TSA in attenuating the oxidative stress in ovarian tissues of aged mice.

F I G U R E 3
Quantitative RT-PCR revealed relative mRNA levels (a) and protein expression of ERK1/2, Cx37, CAT, Smad5, Nrf2, and GPX1 in ovarian tissues of young (4 weeks old) (b) and aged (40 weeks old) (c) female mice after administration of different doses of tanshinone IIA (TSA) (0, 10, 20, 40 μg/g/day). The expression of these mRNAs was normalized to the expression level of GAPDH measured in the same RNA preparation. Different letters above the bars (a, b, c, and d) indicate significant differences (p < 0.05)

DISCUSSION
The dried root of S. miltiorrhiza is one of the most used herbs in Chinese medicine because of its high value. Growing evidence indicates that natural compounds such as TSA possess remarkable curative effect and minute adverse reactions. TSA is a major lipophilic component of extracts from the root of S. miltiorrhiza. It has anti-inflammatory activity (Jang et al., 2003), antioxidant (Fu et al., 2007), cytotoxic to various cancer cell (Chiu & Su, 2010). Also, it has traditionally been used in the treatment of cardiovascular diseases (Luo et al., 2015) through its F I G U R E 4 An overall scheme showing the mechanisms by which tanshinone IIA (TSA) enhances the ovarian reserve in aged mice antioxidant effect (J. Zhu et al., 2017). More importantly, TSA is a new candidate target to be used as an anti-tumour drug in clinical practice and contributes to the suppression of ovarian cancer growth (Zhou, Sui, et al., 2020). TSA has also shown promising results pertaining to treatment of polycystic ovarian syndrome in mice (Jin et al., 2019). Studies to identify the molecular and cellular mechanisms will further shed light on the importance of TSA both for the development and function of reproductive tissues as well as disease condition.
Previous studies have shown that oxidative stress resulting from aging declines egg quality and ovarian reserve as well, which has a serious impact in the maintenance of female fertility (May-Panloup et al., 2016;Yang et al., 2021). Thus, alleviating oxidative stress in the ovaries is an important step for delaying ovarian aging. Nrf2 is an important transcription factor for cell self-antioxidation and plays a major role in controlling the cellular susceptibility to reactive oxygen species (ROS)-induced cytotoxicity (H. Zhu et al., 2005). Endogenous intracellular antioxidant enzymes provide the primary defence against oxidative stress caused by the accumulation of free radicals. GPX1 and CAT are the most common enzymatic antioxidants and play critical roles in removing the harmful oxygen products produced by superoxide dismutase (Fridovich, 1995). For this reason, equilibration status of oxidative stress is very essential process in reproductive medicine (Akino et al., 2018). High expression of Nrf2 in aged mice after administration of TSA might lead to alleviate oxidative stress in ovarian tissue, and this could provide novel insight to conquer the age-related fertility decline that is mainly attributed to the accumulation of aberrant oxidative stress. Moreover, it has been investigated that total apoptosis rates were significantly increased in women with diminished ovarian reserve when compared to women with normal ovarian reserve, along with lower AMH level (Y. Fan et al., 2019).
Nrf2 is critically involved in the regulation of cell proliferation and apoptosis during the decline of ovarian reserve (Chen et al., 2017). The primary endogenous antioxidant defence enzymes, CAT, and GPX1 are together responsible for protection against the damaging effects of free radicals. In this regard, antioxidants from natural products are recommended because they have a high anti-oxidative stress capacity and are safe and acceptable compared with synthetic antioxidants (Yang et al., 2021). Since ovarian reserve is an important factor in achieving pregnancy both in natural and assisted reproduction, TSA-mediated regulation of antioxidants of the ovarian tissue is of importance for protection against ovarian oxidative disorders (Figure 4).
A similar pattern was observed in the aged group is that the levels of gonadotropins level (FSH and LH) increased, whereas the levels of E2 and AMH decreased, indicating a diminished ovarian reserve. In support of our findings, it has been demonstrated that elevated FSH and LH is closely related to the decrease of ovarian function (Takeo et al., 2012). However, in aged mice, administration of medium and high doses of TSA significantly reduced the level of FSH and LH, and increased the levels of E2 and AMH, suggesting that TSA can effectively alleviate the adverse effect of ovarian aging. Furthermore, AMH is a glycoprotein of the TGFβ superfamily, and is suggested as the most specific available marker for ovarian reserve (Kaya et al., 2010;Visser et al., 2012) was found low in aged mice. One of the roles of AMH is to inhibit primordial follicle activation, which slows the rate at which the ovarian reserve is depleted. Elevated AMH level after administration of TSA in aged mice might be one possibility by which TSA promotes the ovarian reserve, but further studies will be necessary to determine the mechanism by which this is achieved.
ERK1/2 and Smad5 family proteins are widely expressed in oocytes and granulosa cells at various developmental stages (Heldin & Moustakas, 2012;Sakaki-Yumoto et al., 2013) and are very essential for ovarian functions. ERK1/2 are necessary for LH-induced oocyte resumption of meiosis, ovulation, and luteinization (H.-Y. Fan et al., 2009). Moreover, defect in Smad5 family proteins expression is known to have adverse effects on ovarian organogenesis and folliculogenesis (Kaivooja et al., 2006). The expression level of these genes was significantly decreased in aged mice; however, administration of TSA particularly with medium and high doses improved the expression levels of ERK1/2 and Smad5 family at both mRNA and protein levels, suggesting that TSA maintains the ovarian functions through different ways.
Connexin 37 is critical to ovarian function as it participates in sustaining proper growth and maturation of the oocyte and plays an important role in folliculogenesis (Teilmann, 2005). A targeted mutation of Cx37 induces female infertility (Gittens & Kidder, 2005). Similarly, in sheep ovary, it plays a role in follicular development and ovulation (Borowczyk et al., 2006). In this regard, we infer that aging has an adverse effect on the expression of Cx37 which may explain, at least in part, the low fertility of aged female mammals. The high expression of Cx37 at both mRNA and protein levels after TSA administration could inspire relevant researches for its potential medicinal value.
Taken together, these findings argue for a supportive role of TSA during ovarian ageing in female mammals that may be of value clinically.
The reasons for the positive effect of TSA on ovarian reserve of aged but not in young mice remain unclear and require further investigation.
However, the higher accumulation of ROS of the aged mice could render them in an oxidative stress that is diminished by the TSA administration. In

CONFLICT OF INTEREST
The authors declare no conflict of interest.

ETHICS STATEMENT
All animal procedures used in this study were carried out in accordance with the Guide for Care and Use of Laboratory Animals (8th edition, released by the National Research Council, USA) and were approved by the Institutional Animal Care and Use Committee (IACUC) of Guangxi

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.